Human monocytotropic ehrlichiosis—A systematic review and analysis of the literature

Human monocytotropic ehrlichiosis (HME) is a tick-borne bacterial infection caused by Ehrlichia chaffeensis. Most available data come from case reports, case series and retrospective studies, while prospective studies and clinical trials are largely lacking. To obtain a clearer picture of the currently known epidemiologic distribution, clinical and paraclinical presentation, diagnostic aspects, complications, therapeutic aspects, and outcomes of HME, we systematically reviewed the literature and analyzed and summarized the data. Cases of HME are almost exclusively reported from North America. Human infections due to other (non-chaffeensis) Ehrlichia spp. are rare. HME primarily presents as an unspecific febrile illness (95% of the cases), often accompanied by thrombocytopenia (79.1% of the cases), leukopenia (57.8% of the cases), and abnormal liver function tests (68.1% of the cases). Immunocompromized patients are overrepresented among reviewed HME cases (26.7%), which indicates the role of HME as an opportunistic infection. The incidence of complications is higher in immunocompromized compared to immunocompetent cases, with ARDS (34% vs 19.8%), acute renal failure (34% vs 15.8%), multi organ failure (26% vs 14.9%), and secondary hemophagocytic lymphohistiocytosis (26% vs 14.9%) being the most frequent reported. The overall case fatality is 11.6%, with a significant difference between immunocompetent (9.9%) and immunocompromized (16.3%) cases, and sequelae are rare (4.2% in immunocompetent cases, 2.5% in immunocompromised cases).


Introduction
Ehrlichioses and anaplasmoses are tick-borne zoonotic infections caused by gram-negative, obligate intracellular bacteria of the family Anaplasmataceae in the order Rickettsiales.All members of the Anaplasmataceae have in common that they survive in vacuoles of host cells, which usually originate from bone marrow, but occasionally also from endothelial cells.The different Anaplasmataceae vary with regard to their tropism for certain cell lines.E.g.Ehrlichia chaffeensis, Ehrlichia canis and Ehrlichia muris infect mostly the monocytes and macrophages, while Anaplasma phagocytophilum and Ehrlichia ewingii infect mostly the granulocytes of their mammalian hosts.This tropism for certain cell lines is reflected in some of the historical names of the illnesses these pathogens cause in their natural animal as well as accidental human hosts.In the context of human infection this is human monocytotropic ehrlichiosis (HME) and human granulocytotropic anaplasmosis (HGA).

Human ehrlichiosis
The first recognition of Ehrlichia as a pathogen dates back to 1935, when canine ehrlichiosis caused by Ehrlichia canis and its transmission by Rhipicephalus sanguineus ticks was discovered.Later studies revealed that two other economically important veterinary pathogens, Anaplasma marginale (first described in 1910) and Cowdria ruminantium (first described in 1925), are also ehrlichiae [1].The first human infections caused by Ehrlichia were reported as early as 1954, when Rickettsia sennetsu (later Ehrlichia sennetsu, today Neorickettsia sennetsu) was identified as the causative agent of Sennetsu fever (a glandular fever-like disease) in Japan [2,3].Until then, bacteria of the family Anaplasmataceae (today including the genera Ehrlichia, Anaplasma and Neorickettsia) were primarily known as veterinary pathogens.This changed following the identification of Ehrlichia chaffeensis (then thought to be E. canis) in a blood sample of a 51-year-old man presenting with a febrile illness clinically similar to Rocky Mountain spotted fever after a tick bite in rural Arkansas in 1986 [4,5].Since then, human monocytotropic ehrlichiosis (HME) due to E.chaffeensis has increasingly been recognized as an important tick-borne pathogen in the United States.
In 1999, E. ewingii (first described in 1971 [6]), a Ehrlichia strain closely related to E. canis and E. chaffeensis and, like E. canis a causative agent of canine ehrlichiosis, was found to also cause illness in humans.These E. ewingii infections presented with a clinical picture similar to that of HME caused by E. chaffeensis [7].The natural enzootic cycles of both E. chaffeensis and E. ewingii primarily involve the Lone Star tick (Amblyomma americanum) and the white-tailed deer (Odocoileus virginianus) as vector and reservoir.Human infections represent zoonotic spillover events mostly related to recreational, occupational, military-related or peridomestic exposure to the tick vector [8].In 1999, HME became a reportable disease in the United States.Between 1987 and 2017, 15,527 cases of HME were reported in the USA [9].From 2008 to 2012, the incidence of HME was 3.2 cases per million population in the United States, a fourfold increase from 2000 [10].In areas of high endemicity, incidence rates as high as 138-330 per 100,000 population [11,12] and seroprevalence rates of up to 12.5% are found [11].In 2009, the spectrum of Ehrlichia species causing human ehrlichiosis (HE) in the United States extended to E. muris subsp.eauclairensis (formerly Ehrlichia muris-like agent), whose enzootic cycle primarily involves the blacklegged deer tick (Ixodes scapularis) and the white-footed mouse (Peromyscus leucopus) and other small mammals as vector and reservoir [13].Table 1 shows the currently known human pathogenic Ehrlichia species.
Human ehrlichial infections/pathogens have also been reported in South America, Africa, and Asia, but the epidemiology of HE outside the United States is still largely unknown [19][20][21][22][23].
The taxonomic order of the Anaplasmataceae underwent significant changes over time, with the most relevant one being the identification of Anaplasma as an own genus and its separation from the genus Ehrlichia in 2001 [1].With this change of taxonomic order, the previously called human granulocytotropic ehrlichiosis (HGE, first described as a human pathogen presenting similarly to HME in 1994 in the United States [24] became human granulocytotropic anaplasmosis (HGA)).Although human infection caused by all members of the family Anaplasmataceae have been, and often continue to be, generically referred to as ehrlichiosis, it is increasingly apparent that the clinical manifestations and causative agents are distinct [8].

Clinical presentation
Human ehrlichiosis and human anaplasmosis both present as febrile illnesses manifesting one to two weeks after the bite of an infected tick.The fever is accompanied by unspecific symptoms like weakness, malaise, headache, myalgia, arthralgia, nausea, and vomiting and sometimes a rash is present.Most infections are uncomplicated and self-limiting (and most likely even asymptomatic), but severe complications, including septic shock, acute respiratory failure, meningoencephalitis, renal failure, and multi-organ failure may occur and even be fatal.The higher incidence and risk for life-threatening complications in older patients suggests that host factors are important parameters influencing disease severity.Increased severity and mortality is also observed in immunosuppressed or immunocompromised patients, particularly in HME.

Diagnostics
Diagnosis of HME and HGA rests primarily on clinical suspicion, epidemiological plausibility, and suggestive unspecific routine blood laboratory results, as the availability of sensitive and specific acute diagnostic tests (i.e.PCR) is often limited.Since the risk for complications increases and prognosis worsens if treatment is delayed, it is recommended to start empirical antimicrobial treatment before the results of the laboratory tests are available [8].Routine blood laboratory investigations often show cytopenia, particularly leukopenia and thrombocytopenia, elevated liver function test levels (transaminases, alkaline phosphatase), and elevated C-reactive protein levels [25].
Light microscopic examination of blood smears (stained with e.g.Wright's, Diff-Quik, Giemsa) may be diagnostic if the typical, intracellularly located inclusions called morulae (from the Latin word for mulberry) are detected.These morulae represent clustered microcolonies of the pathogen within the host cell vacuole (Fig 1).Although this method is rapid, it is relatively insensitive compared to other confirmatory tests, especially beyond the acute phase (first week of infection) and in immunocompetent patients where disease is usually associated with a very low bacterial burden [8,25].The sensitivity of microscopy also largely depends on the causative species: morulae are detectable in 25-75% of the blood smear examinations in HGA but only in 3-7% of the blood smear examinations in HME [8,25].Except for microscopy, PCR assays, serology, immunostaining of biopsy/autopsy material, and cell culture are used to diagnose HME and HGA.Table 2 gives an overview on the advantages, disadvantages and applications of the different diagnostic methods available.

Treatment and outcome
The treatment of choice for human ehrlichiosis and anaplasmosis is doxycycline or tetracycline and in most cases, clinical response is rapid.The observation that mildly affected patients recover spontaneously even without specific therapy, as well as the high seroprevalence rates reported in some populations, suggest that most infections are mild, self-limiting and to a large extend probably subclinical [8,25].
National surveillance data for HME from the United States show a hospitalisation rate of 57% and an overall death rate of 1%.Higher case fatality rates of 4% in children <5 years and 3% in the elderly �70 years are reported [10].
With our systematic review of human ehrlichiosis presented here, we aim to provide clinicians with a comprehensive summary of the available data, focusing on the clinically relevant core aspects (Note that an analogously compiled systematic review of human granulocytotropic anaplasmosis (HGA) was conducted in parallel by our group).

Methods
We performed a systematic literature search of the databases EMBASE, CINAHL, Cochrane, PubMed, Scopus and Web of Science on 04/Jan/2023, using the search term (("Ehrlichia" OR "Ehrlichiosis" AND "Monocytes")) OR ("Ehrlichi*" OR "Neoehrlichi*" OR "HME Agent" OR "chaffeensis" OR "ruminantium" OR "muris eauclairensis" OR "muris" OR "sennetsu" OR "ewingii" OR "Ehrlichia canis") NOT ("Animals" NOT "Humans")), adapted to the search format of the different databases.A detailed description of the literature search is available in S1 Text.After removing duplicated by EndNote (Version X9.3.3) and manually, the publications were pre-screened by title and abstract, removing those not concerning HME or not including the objectives of this study (data of clinical cases, including epidemiology, mode of transmission, diagnostic, treatment, outcome).A full-text review of the remaining publications was then performed excluding those not meeting the inclusion criteria, according to the systematic review protocol (concerning HME and the objectives of the study, published in English, German, French, Italian or Spanish), as prescribed in S2 Text to exclude those not meeting the inclusion criteria.Publications that could neither be retrieved through the respective journals, nor by contacting libraries or the corresponding authors, were classified as 'not retrievable' and excluded.During the full-text review, the reference lists of the publications were screened for additional relevant articles not identified previously («snowball-search» strategy).We conducted a second literature search with the identical search strategy on 26/June/2023 to include the most recently published articles.From the finally screened eligible studies, we extracted the following data: author, title, year of publication, type of study, country of study, study period, number of HME cases reported, age, sex, most likely country/province of acquisition/  3).The grading system was derived from the CDC case definition of HME [23].For interpretation of the laboratory parameters, we used our in-house laboratory reference ranges.For parameters not commonly done in our laboratory, we used the reference ranges of the University Hospital of Basel.The reference table used to interpret the laboratory findings can be found in S3 Text.To limit confounding, patients with coinfections and preexisting conditions were excluded from some of the analyses if it was not clear whether the symptoms, the laboratory abnormalities, complications or the outcome were attributable to HGA.
For analysis, we divided the reviewed clinical cases into two categories, cases reported with individual data (CRID) and cases reported with non-individual data (CRNID), i.e. case series with cumulatively reported/pooled data, and differentiated between HME monoinfections and cases of HME with coinfection(s).The data extraction sheet is available in S4 Text.Data were analyzed and descriptively summarizing in percentages, medians, and ranges.We used the free online geographic application Mapchart (www.mapchart.net)to visualize the worldwide and regional distribution of HME cases and the causative Ehrlichia spp.This review was registered on PROSPERO under the number CRD42022385413.

Results
Our search identified 12,646 publications, of which 264 proved eligible for inclusion in the review (Fig 2).The reference lists of the included and excluded publications, considered publications, and the PRISMA checklist for systematic reviews are available in S5 Text and S6 Text, respectively.
246 of the 264 analyzed publications reported information on 416 CRID.The other 18 analyzed studies reported on cohorts of HME patients with a total of 844 CRNID.For the analysis of signs and symptoms, laboratory findings, complications and outcome, we used data only from cases clearly indicating the causative species as E. canis or E. chaffeensis monoinfections, thus resulting in 321 CRID and 659 CRNID.The analysis of the data on epidemiology, diagnostics and antimicrobial treatment included mono-and coinfections.Although the analysis of the cases reported with non-individual data (CRNID, i.e. case series, cohorts) was limited, we nevertheless summarized the data analogously to CRID to allow a rough overview and comparison (S7 Text).We also summarized the data on CRID with coinfection(s) (S8 Text), but due to the data heterogeneity and the overall low numbers, we omitted the attempt of specific subgroup analyses.Fig 3 shows the allocation of cases to the respective analysis group.
It should be noted that throughout the further manuscript we use the term HME when specifically referring to human infections with E. canis and E. chaffeensis, while we use the term HE when referring to human infections with Ehrlichia, regardless of species.4 show the number of publications reporting cases of human ehrlichiosis, the number of CRID and CRNID, and the geographic origin of these reports from 1987 to 2023.

Epidemiology
Table 5 lists the number of CRID and CRNID reported from the different regions of the world.Fig 5 shows the worldwide distribution of reported HE cases.Fig 6 shows the distribution of HE cases in the USA according to national surveillance data and according to the data analyzed for this review.
Fig 7 shows the number of HE due to E. canis and E. chaffeensis.The red line marks the year in which it was discovered that in humans the culprit Ehrlichia species is E. chaffeensis and not E. canis [4].
Of the 264 analyzed publications, 12 publications reported a total number of 61 non-canis/ non-chaffeensis HE cases (Table 6).In 32 cases of HE, a co-infection was present.The most common co-infection was with Rickettsia spp., the 2 nd most common with Borrelia burgdorferi (Table 7).Data on the diagnostic methods used to diagnose HE was available for 413 CRID and 814 CRNID, the majority of cases having been diagnosed with PCR (Table 8).In 66 cases, the detection of morulae by light microscopy was described.In 43 of these cases (65.2%), the diagnosis of HE was confirmed by PCR and in 32 of the cases the cell line containing the morulae was specified (Table 9).
The use of cell culture was reported in 17 cases.In 15 cases, a DH82 cell line was used [30,41] and in two cases the used cell line was not specified [42,43].
For 111 cases (26.7%), preexisting immunocompromization/-suppression was reported: 92 were on immunosuppressive medication, 61 of them due to a solid organ transplantation; 14 were HIV positive, 10 of them with a reported low CD 4 cell count or AIDS defining conditions; three had a splenectomy; one chronic renal failure; one was described as immunosuppressed by the authors without further explanation.The suspected route of transmission was tick-borne in 245 (96.1%) cases.Among 210 patients actively assessed for the history of a tick bite, 152(72.4%)recalled a tick bite.The culprit tick species was only mentioned in two cases, in both cases it was Amblyomma americanum (the Lone star tick).In 114 (97.4%) of 117 cases with respectively available data, outdoor activities were reported as risk factor for HME.The reported outdoor activities were recreational (camping, hiking, biking, fishing, playing golf, gardening) in 73% and occupational (most commonly work in construction, on farms or military training, rarely field biology) in 27% of the cases.Other reported transmission routes included blood transfusion (n = 3; Table 10) and organ transplantation (n = 7; Table 11).
Two cases of HE related to international travel were reported (Table 12).One case of HE during pregnancy was reported.The woman was 13 weeks pregnant.She recovered completely and both mother and infant showed no long-term sequelae during the 1 year of follow-up.No data on whether vertical transmission occurred was reported [46].For 299 HME monoinfection CRID the information whether the patients were symptomatic or asymptomatic was reported: 298 (99.7%) were symptomatic, 1 (0.3%) was asymptomatic.For 175 (58.7%)HME monoinfection CRID, data on the timespan from symptom onset to presenting to a physician or hospital was available: median 5 days (range 0-28 days).Data on the incubation period in case of tick-borne transmission was available for 72 cases: median 14 days (range 1-66 days).Data on the incubation period in case of transmission by organ transplantation was available for all 7 reported cases: median 11 days (range 9-25 days).Data on the incubation period in case of transmission by blood product transfusion was only available for only one of the 3 reported cases: 22 days (Table 10).
Hospitalization was reported for 248 (83.2%) of the symptomatic cases.Data on the length of hospitalization was available for 76 patients: median eight 8 days (range 1-127).

Signs and symptoms
For 290 of the 299 HME monoinfection CRID, data on signs and symptoms was available (Fig 3).Fig 8 shows the frequency of the most commonly reported symptoms.Information on the exact duration of fever was available for 39 patients: median 8 days (range 1-120), with five patients having a persistent or recurring fever for more than 20 days.For 153 cases, data on the maximum temperature measured was available: median 39.4˚C (range 36.3-41.6˚C).

Laboratory findings
Of the 299 HME monoinfection CRID, data on laboratory findings was available for 254 cases (Fig 3).Fig 9 shows the frequency and Table 13 the median values and ranges of the reported abnormal laboratory findings.underlying medical condition was not considered a complication of HME (e.g.exacerbation of COPD).Hemophagocytic lymphohistiocytosis (HLH) was reported in 64 cases: 43 HME monoinfection CRID and 21 non-canis/non-chaffeensis HE cases and/or in cases with coinfection (Table 14).

Treatment
Data on antimicrobial treatment was available for 396 of the 416 analyzed HE CRID: 381 cases (96.2%) received antimicrobial treatment, 11 (2.8%) cases received no antimicrobial treatment, and in four cases (1%) the antimicrobial treatment was not specified.Of the 381 cases receiving antimicrobial treatment, 365 (95.8%) received appropriate antimicrobial treatment (* substances listed in Table 15).One case was treated with tigecycline, which was considered adequate for the treatment of HE by the authors [18].
For 88 of the 365 appropriately treated patients, the time between presentation to a physician or hospital and administration of appropriate therapy was known: median 2 days (range 0-38 days).In 235 of the 365 appropriately treated cases (64.4%), antimicrobial treatment was initiated empirically, in 37 cases (10.1%) antimicrobial treatment was initiated after receiving a positive diagnostic result for HE, and for 93 cases (25.5%), information was not available.Data on the time window between symptom onset and administration of appropriate antimicrobial treatment was available for 62 cases: median 7 days (range 0-59).
Data on the time window between first administration of appropriate antimicrobial treatment and the resolution of fever was available for 72 cases: median time 2 days (range <1-6 days).In one case, treatment was only started after the fever had already subsided.In the majority of cases, doxycycline in the standard dose of 200 mg per day (100mg BID) was given.The overall median duration of antimicrobial treatment was 14 days (range 4-30 days) (Fig 11).

Outcome
Data on the outcome was available for 292 of the HME CRID: 34 (11.6%)died due to acute complications related to their infection (Table 16), 21 of them were immunocompetent, 13 were immunosuppressed, resulting in case fatality rates of 9.9% and 16.3% respectively.Information on the time between first symptoms and death due to ehrlichiosis was reported in 19 cases: median 13 days (range 7-68 days).Of the survivors with respectively available data, 11

Publications on HME
Since HME was first described in 1987, the number of publications reporting cases of HME has increased slightly over time, with large cohort studies published in 1994, 2019 and 2020 each providing data on more than 100 cases (Fig 4) [37,115,116].

Non-vector-borne pathogens
Escherichia coli PLOS NEGLECTED TROPICAL DISEASES

Epidemiology
The vast majority of HE cases are reported from the USA (Table 4 and 5   Survived [17] DIC, disseminated intravascular coagulopathy; DM2, diabetes mellitus type HE or report case series of human infections with unusual Ehrlichia species, like E. ruminantium in South Africa [38], or Candidatus Ehrlichia erythraense in China [40] (Table 6 and paragraph below).Thus, it remains largely unclear if HME and to what extend HE exists outside North America, especially, in the absence of known transmission cycles, vector ticks or animal reservoirs.An unspecified febrile illness may easily be misdiagnosed as ehrlichiosis if based on serology only, as serological assays are hampered by cross-reactivity among Anaplasmataceae species, such as Anaplasma phagocytophilum [10].This might explain some of the putative HE cases reported from regions outside the known ehrlichiosis endemic areas (Fig 5).The latter are well characterized for the US, with our analyzed data nicely mirroring the national surveillance data (Fig 6).

Level of diagnostic certainty of human ehrlichiosis
Coinfections Ref.

Non-canis/non-chaffeensis human Ehrlichiosis
Only a very small proportion of the published cases of HE are due to non-canis/non-chaffeensis Ehrlichia species (Table 6).The most frequently reported non-canis/non-chaffeensis Ehrlichia species in the US is E. ewingii, which accounts for 5.5% of reported HE cases, according to  Heitmann and colleagues [10].In our analysis we found that E. ewingii is reported in 3.3% of the published cases, but it should be pointed out that the proportion of reported E. ewingii cases is likely influenced by the used PCR assay [10].

Coinfections
Among the reviewed HE cases, we found a coinfection rate with other tick-borne pathogens of 2.1% (26/1260) (Table 7).However, reported data on the rate of co-infection in HE cases should generally be interpreted with caution (limited sample sizes, questionable representativeness, lack of systematic testing for coinfections).In addition, the report of concomitantly diagnosed coinfections should especially interpreted with caution if the causative pathogens are exclusively transmitted by different tick species.

Diagnostic
The most frequently reported diagnostic methods used to diagnose HE are PCR and serology (Table 8).PCR is highly specific but specificity varies among assays.In the reviewed publications, often an unspecific Ehrlichia PCR assay was used, which might lead to an overestimation of HE cases, as some of these assays not only detect Ehrlichia spp.but also Anaplasma spp.[117,118].In serology, similar specificity problems arise due to the cross-reactivity of serological assays with pathogens from the Anaplasmataceae family as well as between different Ehrlichia species [21].In addition, false positive serological results may also be seen.A notable example in this regard is that of a woman with clinical symptoms consistent with HE who had a positive E. chaffeensis-specific IgM and IgG test result but did not respond to appropriate antimicrobial treatment.This patient was eventually diagnosed with systemic lupus erythematosus (SLE), and after SLE-specific treatment, her ehrlichiosis serology became negative, confirming that her false-positive test was likely due to SLE [116].The higher proportion of CRID diagnosed by PCR compared to CRNID (Table 8) is probably due to the fact that CRID were more frequently reported in context of acute infection, for which PCR is the ideal tool, whereas CRNID were more frequently reported in retrospective studies and therefore diagnosed retrospectively by serology.Microscopy of blood smears for the presence of morulae is rarely performed.This is probably due to the fact that the presence of morulae in HME is rare overall (much rarer than in anaplasmosis) and therefore the sensitivity is low, and that PCR is a much more sensitive method for acute diagnosis.In connection with microscopy, it should be noted that the tropism of E. chaffeensis for monocytes is not 100%, contrary to the suggestive designation human monocytotropic ehrlichiosis (Table 9).
Immunostaining of biopsy tissue and culture is rarely performed (Table 8), which is understandable, considering that these diagnostic methods are technically challenging as well as resource and time demanding and rarely available.

Patients' characteristics
HME was more commonly diagnosed in males than in females (M:F ratio = 1.84) and mostly seen in middle-aged and older individuals, which corresponds to US surveillance data [119] and most likely reflects the higher recreational and/or occupational exposure risk of males to ticks.Of the cases we analyzed, 26.7% were reported in immunocompromized patients.In 54.9% of these patients the immunocompromization was due to past organ transplantation.This is in complete agreement with the literature, which usually states that a quarter of HME patients fall into the group of immunocompromized patients [8].HME is therefore an opportunistic infection that should be included in the differential diagnoses of immunocompromized patients, particularly organ transplant recipients, living in endemic areas.
Among our reviewed cases, the hospitalization rate was 83.2%, which is considerably higher than the 57% reported in US national surveillance data [10], but similar to the hospitalization rate of 88.4% reported by a tertiary care centre in an endemic area [116].The discrepancy in the reported hospitalization rates is thus plausibly explained by the nature of the underlying data sources with our data primarily reflecting the more likely published data on severe cases.

Route of transmission
The most common route of transmission of HME is by tick bite, which is confirmed by the fact that in 96.1% of the reviewed HME cases the reported suspected route of transmission was   a tick bite.Transmission through blood transfusions (n = 3; Table 10) and organ transplants (n = 7; Table 11) is reported, but appears to be rather rare overall.Whether HE is vertically transmissible in case of infection during pregnancy remains unclear.We encountered only one case report of HE during pregnancy, and in this case no vertical transmission was reported.

Travel-related cases
With only two ever reported cases, HE appears to not be a relevant infection in the context of international travel.Although the awareness of physicians to suspect HE as well as the diagnostic capacities to confirm HE is likely very low outside US endemic areas, we do not consider it likely that HE is frequent among international travelers.

Signs and symptoms
HME presents similarly to many other febrile illnesses without specific signs and symptoms (Fig 8 ) and can be easily misdiagnosed or overlooked as it is often mild, oligosymptomatic and self-limiting, even in the absence of antimicrobial treatment.The majority of ehrlichiosis infections may even be asymptomatic and therefore go unnoticed, as supported by respective case reports [114].Since Ehrlichiae belong to the order Rickettsiae, it is not surprising that the classic clinical triad of fever, headache and skin rash is also observed in HE.However, we found this classical triad reported in only 10% of the reviewed cases.The presence of a rash was described in 24.5% of the reviewed cases, with some discrepancies when looking on certain subgroups: e.g.rash was reported in 43% of pediatric and 19% of adults HME cases and in 28% of immunocompetent and 13% of immunocompromized cases, respectively.The range and difference in the age-specific proportions is roughly in line with the literature, where the presence of a rash is described in approximately 48-60% of pediatric and <30% of adult HME cases [42,120].Regarding our finding of a lower, but still considerable rate of rash in immunocompromized patients, it is interesting that in a retrospective study comparing immunocompetent HME cases (n = 43) with organ transplanted HME cases (n = 15), the only statistically significant different symptom was rash, being present in 38% of immunocompetent but 0% of organ transplant patients [31].

Laboratory findings
Laboratory findings in HME cases are not specific, but often characterized by the combination of cytopenia (with thrombocytopenia being the most common, followed by leukopenia and less commonly mild non-hemolytic anemia) and elevated transaminase levels (Fig 9).However, this pattern may also be observed with other infections, including many other tick-borne infections (e.g., anaplasmosis, rickettsiosis, tularemia, babesiosis, tick-borne relapsing fever, Q fever, and tick-borne arboviral infections) [121].

Complications
The reported rate of complications among the reviewed monoinfected CRID was 63.1% and thus rather high when compared to the complication rate of up 17% according to the literature [25,115,116].This difference is very likely explained by reporting and publication bias, but very likely also by differences in case definition.We defined a complicated course of HE as a case whose severity requires specific therapeutic interventions/care beyond the administration of antimicrobial therapy (e.g., respiratory support, renal replacement therapy, circulatory support, blood transfusion, intensive care monitoring etc.), whereas many publications restricted the definition of complications to life-threatening organ-failure.Also, the results of our analysis regarding complications very likely reflect a worst-case scenario, as the denominator used to calculate the incidence of complications was conservatively chosen when using only data from cases in which data on complications where available.In addition, 25% of the cases with reported complications we reviewed were immunocompromised.As complications are known to occur more frequently in immunocompromised individuals [122][123][124][125], this may also have contributed to the higher complication rate we found.
The most common complications were ARDS, acute renal failure, multi-organ failure and HLH, with the first two complications occurring statistically significantly more frequently in immunocompromized patients ( Fig 10).Particularly HLH appears to be a prominent complication of HE.HLH comes in two forms, primary (inherited) HLH, and secondary (acquired) HLH (sHLH).The latter has many known triggers, including neoplasms, autoimmune processes, and particularly infections, with the most common infectious cause being viral [85,126].sHLH results from defective hyperactivation of natural killer and cytotoxic T-cells that can progress to multi-organ failure [126].In septic patients, the incidence of sHLH is estimated between 3.7 and 4.3% [126].Among the reviewed cases with reported data on complications, HLH was reported in 17% (43/252) of the cases.This number is very likely overestimating the frequency of HE-related HLH due to reporting and publication bias, but even if assuming the absence of HLH in all reviewed HE-cases not specifically reporting the absence of HLH, the incidence of HLH would still be 6.5% (64/980).Although many pathogens are known as possible triggers of HLH, we have not found any other pathogen in the literature that has a similarly high association rate with HLH as Ehrlichia.We found a CFR among HE-related HLH cases of 18% (12/64).Since the overall reported case fatality rate of HLH is 50-80% [127], HE-related HLH appears to have a comparatively good outcome.

Treatment
All patients with a suspected diagnosis of HME should be treated empirically without delay, regardless of whether the diagnosis is confirmed, as delayed treatment leads to prolonged illness, longer hospitalization, and an increased risk of complications [128].Doxycycline is the most commonly and widely used antimicrobial agent (Table 16) and the recommended treatment of choice.The most frequently recommended and most widely adopted dosing regimen for adults is 100mg of doxycycline twice daily [26], but no studies have yet been conducted to define an optimal treatment regimen for HME.In our analysis, the median duration of antimicrobial treatment was 14 days, with a clear tendency of physicians to either opt for a 7-, 10-, 14-or 21-day treatment regimens ( Fig 11).The tendency to opt for 10-14 days is probably due to the fact that physicians want possible Lyme co-infection to be covered [42,129], while for HME, a treatment duration of 4-5 days (corresponding to three days after defeverence) is otherwise considered adequate [26,130].Our analysis confirms that fever resolves rapidly (mostly within 2 day) after starting appropriate antimicrobial treatment.Thus, in the case of persisting fever, the diagnosis of HME should be reconsidered.While tetracycline is contraindicated in children <8 years of age due to the risk of irreversible dental staining, administration of doxycycline for up to 21 days is considered safe at any age [131][132][133].The recommended pediatric dose of doxycycline for children weighing less than 45 kg is 2.2mg/kg body weight twice daily [26].Regarding pregnant woman, doxycycline has shown no evidence for increased teratogenicity [133] and the US CDC states: "in cases of life-threatening allergies to doxycycline, severe doxycycline intolerance, and in some pregnant patients for whom the clinical course of ehrlichiosis appears mild, physicians might consider alternate antibiotics" [130].Of note, with regard to severe doxycycline allergies, a protocol for rapid desensitization to doxycycline has been published [134].Besides doxycycline (or tetracycline), rifampin is the only potentially effective alternative, although there is little data available on it.Rifampin appears effective against E. chaffeensis in vitro [135] whereas animal model data on the efficacy of rifampicin against E. canis is inconsistent [136].The successful use of rifampin in HGA is reported [137,138], but respective reports on its use in children and pregnant women with HME are lacking.Only one case report has been published describing a successful use of rifampin for treatment of HME [139].In addition, rifampin would not be effective in the case of Rocky Mountain Spotted Fever (RMSF), a feared differential diagnosis of ehrlichiosis.The latter is particularly relevant as their endemic areas in the USA overlap considerably.In vitro data show that chloramphenicol, ciprofloxacin, cotrimoxazole (trimethoprim/sulfamethoxazole), erythromycin, penicillin, and gentamicin are not effective against E. chaffeensis [135].

Outcome
Most patients recover without sequelae.In only 4% of cases with data on outcome, sequelae were reported, mostly lasting for a few weeks after acute disease (Table 17).The overall case fatality rate in our analysis was 11.6%, with a higher CFR in immunocompromised than in immunocompetent patients (16.5% vs 9.9%).In the literature, the case fatality rate of ehrlichiosis is much lower.The highest case fatality rate is seen in children <4 years and in patients >70 years with 4% and 3%, respectively [10].The higher case fatality rate in our analysis is likely explained by publication bias as cases with complications and/or fatal outcome are much more likely to be published than mild or asymptomatic cases (Table 16).Regarding immunity after infection with Ehrlichia, there is no conclusive evidence whether patients are susceptible to reinfection or not [122].A single case of molecular proven reinfection by another E. chaffeensis strain has been documented in a liver transplant patient [140].Of note, one case of a despite appropriate antimicrobial treatment (tetracycline and chloramphenicol) persistent and finally fatal infection with E. chaffeensis has been reported and suggests that Ehrlichia, similarly to other intracellular pathogens (like e.g.Coxiella) may, albeit rarely, cause chronic persistent infection [88].

Limitations
Our analyses have several limitations.Next to publication bias and the retrospective nature of most available data sets, most reviewed studies did only provide incomplete data sets.In addition to the inhomogeneity and incompleteness of the available data sets, the data and results of studies that reported on case series or cohorts were often given as total values, medians or percentages, so that it was often not possible to assign the data to individual cases (see section on CRNID).Even though we tried to eliminate case duplicates, the cohort studies we looked at could potentially contain duplicate cases already described in other cohort studies or case reports.When we compare our findings in case studies with the cohorts we looked at as well as CDC data on HME in the USA [119], we see that the course of illness in our cases was comparatively severe, which points to reporting and publication bias.The latter particularly impacts systematic review being based on a considerable number of case reports, as severe cases are more likely to be reported and published than mild cases.
Box 1 summarizes the main conclusions we have drawn from our review and Box 2 contains our selection of publications that we recommend clinicians to read.

Fig 4 and Table
Fig 4 and Table4show the number of publications reporting cases of human ehrlichiosis, the number of CRID and CRNID, and the geographic origin of these reports from 1987 to 2023.Table5lists the number of CRID and CRNID reported from the different regions of the world.Fig 5 shows the worldwide distribution of reported HE cases.Fig 6 shows the distribution of HE cases in the USA according to national surveillance data and according to the data analyzed for this review.Fig7shows the number of HE due to E. canis and E. chaffeensis.The red line marks the year in which it was discovered that in humans the culprit Ehrlichia species is E. chaffeensis and not E. canis[4].Of the 264 analyzed publications, 12 publications reported a total number of 61 non-canis/ non-chaffeensis HE cases (Table6).In 32 cases of HE, a co-infection was present.The most common co-infection was with Rickettsia spp., the 2 nd most common with Borrelia burgdorferi

Table 2 . Overview of laboratory methods available to diagnose HME and HGA and their advantages, disadvantages and application/use [26,27].
or imported case, if imported: time between end of trip and symptoms, country/province of diagnosis, risk factors for tick bite, year of acquisition/infection, pre-existing conditions, immunosuppression, pregnancy, symptomatic/asymptomatic infection, hospital admission, time between first symptoms and presentation to hospital/physician, duration of hospital stay, signs and symptoms, duration between fever and appropriate treatment, presumed vector of disease, history of tick bite/exposure, tick species, duration between bite and symptoms, diagnostic methods (serology, PCR, microscopy, culture, biopsy), grade of diagnostic certainty, Ehrlichia species, coinfections, laboratory data, used drug(s) and treatment regimen(s), number of treated or untreated patients, complications and outcome.Discrepancies and unclear cases were discussed and resolved by consulting a second reviewer.The probability of diagnostic certainty of the individual HME cases was graded according to the diagnostic method used in the included studies, with PCR, culture and immunostaining of tissue having the highest (grade A+) and clinical diagnosis the lowest (grade D) evidence for a correct diagnosis (Table IFA, immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay, PCR, polymerase chain reaction.https://doi.org/10.1371/journal.pntd.0012377.t002infection, autochthonous

Table 6 .
[35]tinued) LDH, lactate dehydrogenase; N.r., none/not reported; PCR, polymerase chain reaction; Ref., reference; USA, United States of America.*regarding[35]:this publication describes an additional five cases of human Ehrlichia ewingii infections.Those cases were not included in our analysis because of insufficient clinical data.

Table 7 . Reported coinfections in human ehrlichiosis (n = 32).
Very few publications report HE cases from regions of the world other than North America.Most of these cases are based on low diagnostic certainty regarding the correct diagnosis of ).Until 1991, it was assumed that cases of HME in the US were exclusively caused by E. canis.Then, in 1991, Anderson et al. identified a new Ehrlichia species in the US, closely related to but different from E. canis, which was named E. chaffeensis.Since then, the majority of published HME cases is reported to be due to E. chaffeensis, with only very few publications reporting cases due to E. canis.This led to the recognition that the main causative pathogen of HME is E. chaffeensis and and not E. canis[4].This paradigm change is nicely illustrated inFig 4.

Table 8 . Diagnostic methods used to diagnose human ehrlichiosis. Diagnostic method Level of diagnostic certainty* Human ehrlichiosis CRID [n = 413] Human ehrlichiosis CRNID [n = 814] Number of cases tested positive by this method n (%) # Number of cases for which this level of diagnostic certainty was the highest n (%) Number of cases tested positive by this method n (%) # Number of cases for which this level of diagnostic certainty was the highest n (%)
CRID, cases reported with individual data; CRNID, cases reported with non-individual data; ELISA, enzyme-linked immunosorbent assay; IFA, immunofluorescence assay; PCR, polymerase chain reaction.* A+, diagnosed by PCR, culture and/or immunostaining of biopsy/autopsy tissue; A, diagnosed by paired IgG IFA serology; B+, diagnosed by microscopy of blood smear or buffy coat preparation; B, diagnosed by single IgG IFA or ELISA serology; C, diagnosed by IgM serology and/or unspecified serology method; D, clinically diagnosed.# in many cases a combination of diagnostic tests was used to establish the diagnosis.Thus, the number of positive test results exceeds the number of cases.https://doi.org/10.1371/journal.pntd.0012377.t008